Abstract 4155: Catabolism of extracellular glutathione supplies amino acids to support tumor growth

Abstract Restricting amino acids from tumors is an emerging therapeutic strategy with significant promise. While typically considered an intracellular antioxidant with tumor-promoting capabilities, glutathione (GSH) is a tripeptide composed of cysteine, glutamate, and glycine that can be catabolized, yielding amino acids that could support tumor metabolism. However, the extent to which GSH-derived amino acids are essential to cancers is unclear. Here, we find that catabolism of extracellular GSH provides critical support for tumors by supplying cysteine. We show that disrupting the tumor’s ability to synthesize intracellular GSH does not perturb its growth, highlighting the potential importance of extracellular GSH. Notably, we find GSH to be abundant in the tumor interstitial fluid, the primary environment surrounding cancer cells in vivo, where it far exceeds cystine levels. Our findings reveal that cancer cell survival and growth can be restored in cystine-deprived conditions through GSH supplementation. This process was dependent on the catabolic activity of γ-glutamyl-transferases (GGT) enzymes, which can break down GSH, yielding glutamate and cysteinylglycine, which can be further brown down to cysteine and glycine. GGT1 overexpression further facilitates this rescue by lowering the amount of GSH required for cell survival. Metabolomic analysis shows that GSH catabolism replenishes cysteine-related products depleted by cystine deprivation. Additionally, pharmacological screening revealed that when cancer cells acquire their cysteine from GGT-mediated GSH breakdown, they become resistant to inhibitors of cystine uptake and utilization. Finally, the systemic treatment of mice bearing breast tumor xenografts with a potent GGT inhibitor prevented circulating GSH breakdown, reduced tumor cysteine levels, and slowed down tumor growth. This effect was reversed by restoring cysteine levels with N-acetyl-cysteine, indicating that cysteine depletion drives the observed reduction in tumor growth. Our findings indicate a non-canonical role for GSH in supporting tumors by acting as a reservoir of amino acids. Depriving tumors of extracellular GSH or inhibiting its breakdown represents a potentially tractable therapeutic approach for cancer patients, as it could restrict tumors' access to critical amino acids. These findings offer a mechanistic explanation for the pro-tumorigenic role of GSH and reshape our understanding of how amino acids, including cysteine, are supplied to cancer cells, unveiling a novel therapeutic avenue for targeting cancer metabolism. Citation Format: Fabio Hecht, Marco Zocchi, Emily Tuttle, Nathan Ward, Bradley Smith, Yun P. Kang, Juliana Cazarin, Zamira G. Soares, Mete Emir Ozgurses, Huiping Zhao, Colin Sheehan, Fatemeh Alimohammadi, Lila Munger, Dhvani Trivedi, Gloria Asantewaa, Sara Blick-Nitko, Jason Zoeller, Ying Chen, Vasilis Vasiliou, Bradley M. Turner, Alexander Muir, Jonathan L. Coloff, Joshua Munger, Gina DeNicola, Isaac Harris. Catabolism of extracellular glutathione supplies amino acids to support tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4155.